A chemical procedure for the quantitation of three pools of one-carbon-substituted folates (Pool 1 comprising 5,10 methylene tetrahydro folates and unsubstituted tetra and dihydro folates; Pool 2 made up of 5-methyl-tetrahydro folates and Pool 3 made up of the folates with one-carbon-substituents at the formyl level of oxidation; 5,10 methenyl tetrahydro folates, 5 and 10-formyl tetrahydro folates and 5-formimino tetrahydro folates) has been developed. The method allows for the determination of the chain length of the poly-gamma-glutamyl peptide corresponding to each of the three one-carbon-subsituted pools mentioned above. This new methology will be applied to study the hypothesis that alterations in the chain length of the poly-gamma-glutamates play a role in the regulation of one-carbon metabolism.